Purpose:
In a previous study, one-week storage of cultured human oral keratinocytes (HOK) was found to be superior at 12°C compared to 4°C and 37°C with regard to viability and morphology. To better understand the processes involved during storage at these three temperatures, we assessed the gene expression of cultured cells before and after storage. The feasibility of storage of cultured HOK is important as it allows for transportation of cultured transplants to eye clinics worldwide.

Methods:
Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and imported into the Partek Genomics Suite software (Partek Inc., MO). Differentially expressed genes (fold change>1,5 and p<0,05) were identified by one-way ANOVA.

Results:
Gene expression in cell cultures stored at 4°C and 12 °C clustered close to the cultures that had not been subject to storage (the control group). Cultures stored at 37°C displayed substantial changes in gene expression compared to the other groups. In comparison with cultures stored at 12 °C, 3534 genes were differentially expressed in cultures stored at 37°C. In contrast, only 153 genes were differentially expressed between the control and the cells stored at 12°C. Compared to cultures stored at 12°C, keratin 10, involved in the differentiation of HOK, was 46-fold upregulated in cultures stored at 37°C. As expected, the putative stem cell marker p63 was down-regulated (~ 2 fold) in cultures stored at 37°C compared to 12°C. Moreover, the heat shock protein encoding gene HspB8 was 28-fold upregulated in cultures stored at 37°C compared to 12 °C, which may indicate cell stress.

Conclusions:
We conclude that 37°C is a suboptimal temperature for storage of HOK in contrast to the current practice of using 37°C as the standard incubation temperature for cell culture.